The culturing of eukaryotic cells, for example mammalian cells, has become a routine procedure and cell culture conditions which allow cells to proliferate, differentiate and function are well defined. Typically, cell culture of mammalian cells requires a sterile vessel, usually manufactured from plastics (typically polystyrene), defined growth medium and, in some examples, feeder cells and serum. The feeder cells function to provide signals which stimulate cell proliferation and/or maintain cells in an undifferentiated state and can influence cell function. The culturing of mammalian cells has many applications and there are numerous in vitro assays and models where cell culture is used for experimentation and research; for example the use of cells in tissue engineering; the use of mammalian expression systems for the production of recombinant protein and the use of mammalian cells in the initial screening of drugs.
Mammalian cells are used in initial drug screening to determine whether a lead therapeutic (e.g. a small molecule agonist or antagonist, a monoclonal antibody, peptide therapeutic, nucleic acid aptamer, small inhibitory RNA) has efficacy before animal trials are undertaken. There is a need to provide improved cell culture systems in which mammalian cells can be cultured to provide a population of cells that are as far as technically possible close to their natural state to enable the analysis of cell proliferation, differentiation and function in a reliable manner.
Cell culture systems are known in the art and have been available to the skilled person for many years. For example, WO2010/005995 discloses micro-patterned co-culture systems as infectious disease analysis platforms. WO2003/014334 discloses an in vitro cell culture method which provides a culture regimen that allows prostate epithelial cells to form 3D prostate-like-acini which closely resemble prostate acini found in vivo. Furthermore, cell culture substrates are described in WO00/34454 which comprises microcellular polymeric materials. These polymers form reticulate structures of pores that interconnect with one another to provide a substrate to which cells can attach and proliferate.
Cell culture typically involves the growth of cells in monolayer culture under sterile conditions in closed cell culture vessels. An approach is to use either 2D protein patterning or 3D micro-wells. 2D protein patterning has been successfully and extensively used to control cell behaviour. In addition micro-well fabrication using silicon etched Si wafers [1, 2], glass [3], polydimethylsiloxane [PDMS] [4], elastomeric stamps [5], hydrogels such as agarose or PEG [6, 7], collagen [8] or Parylen films [9] and fibers [10] have been used for culturing single cells or a limited number of cells in a confined environment. However, the technique usually amounts to using the microwells as miniaturized petri dishes without taking advantage of the confinement of the cells in the well and the possibility of creating 3D protein coated niches. This is mostly due to the fact that chemical coating cannot be different on the sides, top and bottom of the well and can thus only provide a single chemical cue to the cells with little observed advantages over 2D patterned surfaces, at least for single cell culture. In addition, single-cell micro-well technology is not so suitable for high resolution imaging since it suffers from the image distortion created by the index change between the cell and the material used in formation of the micro-wells (e.g. a silica polymer have a refractive index around 1.45) [11].
The present disclosure is illustrated, by example but not by limitation, using primary hepatocytes. Our 3D differential surface coating allows the culture of primary hepatocytes as cell doublets with controlled bile canaliculi [BC] morphology useful in the analysis of drug metabolism. Our technique allows the culture of functional primary hepatocytes at the doublet level for at least 96 hours. In addition our technique offers a versatile standalone membrane that can be included in any standard cell culture device. The cell culture system herein disclosed can be applied to mammalian cells to provide a means to produce cell cultures that mirror more closely in vivo conditions providing a more reliable cell culture system that has applications, for example in tissue engineering, recombinant protein production and drug screening.